Our long term goal is to characterize the mechanisms involved in the regulation of ion transport in tight epithelia. One immediate aim is to analyze the mechanisms that regulate K+ movements across apical and basolateral membranes. It is becoming clear that elucidation of the regulation of K+ conductance is necessary for a full understanding of the signals involved in regulating transepithelial ion transport. We are examining the response of basolateral K+ conductance to increases in cell cyclic AMP induced directly by the addition of cAMP analogs or by neurohypophysial hormones. We are also interested in the modulations of basolateral membrane K+ conductance that occur when transepithelial Na+ transport is either stimulated or inhibited by mechanisms that presumably do not involve changes in cAMP levels. We intend to use conventional microelectrode and fluctuation analysis techniques, as well as the patch clamp technique, to characterize the properties of different K+ pathways. This approach will allow us to determine the relationship between the behavior observed in the whole tissue and that of the isolated channel. By comparing the properties of apical and basolateral K+ pathways we will examine whether or not the same channel is located in both membranes. Another major aim is to study the properties of a cAMP activated, Ca2+-sensitive, monovalent-cation selective channel that we have identified in the apical membrane of the toad urinary bladder and skin. We will examine whether this is a channel akin to the divalent cation channels of excitable tissues and what its role is in the response of epithelia to agents that increase cAMP. Finally, we intend to continue examining the mechanism of the stimulation of Na+ transport caused by hypotonic solutions. This is important because cell volume may be one of the signals involved in adjusting basolateral conductance to changes in ionic flux through the basolateral membrane, and because changes in cell volume trigger volume regulatory responses in cells. We are particularly interested in determining whether hypotonic solutions stimulate cAMP synthesis in the epithelium and the characteristics of their activation of the apical Na+ channel.